Biological characteristics of the nodule-like alveolar lesions (NLAL) induced by 7,12-dimethylbenza(a)anthracene (DMBA) in culture of the whole mammary organ of BALB/c female mice were reported. Fragments of tissues from NLAL areas of the gland, after transplantation into gland-free mammary fat pads of syngeneic virgin hosts, produce hyperplastic lobuloalveolar outgrowths. The mammary hyperplasias (MH) are serially transplantable. Transplantation of dissociated (enzymatic) cells from the MH produced mammary carcinomas. Transplantation of dissociated cells prepared from lobuloalveolar glands, treated with DMBA, also produced mammary hyperplasias and 16/20 (80%) subsequently produced mammary carcinomas. This demonstrates accomplishment of neoplastic transformation of epithelial cell in culture of the whole organ. Twelve mammary hyperplasia lines, derived from mammmary cells transformed by DMBA in the glands in vitro, have been maintained by serial mammary fat pad transplantation for 20 generations. Each of these hyperplasias were tumorigenic, although some failed to show mammary tumors until after 3-6 transplant generations. At each transplant generation transformed cells initially produced MH outgrowths and mammary tumors then appeared in the MH tissue. Findings thus mimic the multistep appearance of mammary tumors, observed in the mouse in vivo. Expression of the casein gene measured by a specific cDNA probe in the hyperplasias showed an altered response to the lactogenic hormone environment of the host mice. Mammary tumors derived from MH tissue in lactating hosts failed to show any expression of the casein gene. The mammary hyperplasias also can grow filling in the whole mammary fat pad in vitro. MH-lines grown in vitro are responsive to the mitogenic action of prolactin, but not to adrenal and/or ovarian steroids. Studies are underway to further characterize the hormone responses of the MH lines in organ culture.